Sulfotransferase, peptide thereof and dna encoding the same

ABSTRACT

A glycosaminoglycan sulfotransferase, a peptide thereof, a nucleic acid comprising a nucleotide sequence encoding the same, an enzyme agent for the synthesis of a glycosaminoglycan, which comprises the above-described enzyme or polypeptide, and a process for producing a glycosaminoglycan, which uses the enzyme agent.

TECHNICAL FIELD

The present invention relates to a glycosaminoglycan sulfotransferase, apeptide thereof, a nucleic acid comprising a nucleotide sequenceencoding the same, an enzyme agent for the synthesis of aglycosaminoglycan, which comprises the above-described enzyme orpolypeptide, and a process for producing a glycosaminoglycan, which usesthe enzyme agent.

BACKGROUND OF THE INVENTION

Unless otherwise indicated, all of the saccharides and saccharideresidues in this description are the D-form optical isomer, excludingiduronic acid. Also, D-glucosamine (which sometimes includes anN-substituted compound) is sometimes referred to as “GlcN”, andN-acetyl-D-glucosamine is sometimes referred to as “GlcNAc”,D-glucuronic acid is sometimes referred to as “GlcA”, L-iduronic acid issometimes referred to as “IdoA”, and hexuronic acid representing auronic acid having 6 carbon atoms, including GlcA and IdoA, is sometimesreferred to as “HexA”.

Heparin and heparan sulfate are kinds of glycosaminoglycan having arepeating structure of a disaccharide (4GlcAβ1/IdoAα1→4GlcNAcα1) of HexAresidue (GlcA residue or IdoA residue) and GlcNAc residue as the basalskeleton (this basal skeleton may be also referred to as “heparinskeleton” hereinafter), wherein one or more of the 2-position hydroxylgroup of its HexA residue and the 2-position amino group, the 3-positionhydroxyl group and the 6-position hydroxyl group of its GlcN residue aresulfated.

It has been known so far that the sulfated group of “heparin” or“heparan sulfate” is bound to one or more of the positions shown by R₁,R₂, R₃ and R₄ in the following formula (2). However, it has not beenknown about a glycosaminoglycan in which all of the R₁, R₂, R₃ and R₄are the sulfate group (SO₃ ⁻) and a production method thereof.

On the other hand it is generally known that a glycosaminoglycan havingthe heparin skeleton has various physiological activities. For example,it is known for a long time that heparin shows anticoagulation activityupon blood (Thronb. Res., 75, 1-32 (1994)), and it is known also that ithas affinity for various growth factors and carries out a role instabilizing or activating these growth factors (Glycobiology, 4, 451(1994)). It is known that heparan sulfate also has affinity for variousgrowth factors and accelerates wound healing by stabilizing oractivating these growth factors (J. Phthol, 183, 251-252 (1997)). Inaddition, it is known that a 6-O-desulfated heparin which can beobtained by specifically desulfating only the sulfate group bound to the6-position of GlcN as a constituting saccharide of heparin is deprivedof the anticoagulation activity upon blood but has an action toaccelerate wound healing (International Publication WO00/06608), and itis known that a periodic acid oxidation-reduction 2-O-desulfated heparin(mainly keeps the heparin skeleton) which can be obtained by acombination of a periodic acid oxidation-reduction treatment andspecific desulfation of the 2-position HexA takes a role in stabilizingvarious growth factors and accelerating nerve growth (JP-A-11-310602).

Based on these facts, it is considered that the glycosaminoglycan havinga heparin skeleton has various physiological activities, and it isconsidered that derivatives of heparin have markedly largepossibilities.

On the other hand, since the gene encoding a glycosaminoglycansulfotransferase has been cloned, it is considered that information onthe substrate specificity of the enzyme for glycosaminoglycan as thesulfate group acceptor can be obtained by preparing the enzyme in alarge amount, which will provide a useful approach in studyingrelationship between the structure and the function ofglycosaminoglycan. It is known that there are many sulfation processesin the synthesis of glycosaminoglycan, particularly in the synthesis ofheparin/heparan sulfate (Glycotechnology, (5), 57 (1994), published byKodansha Scientific), and it is considered that various types ofglycosaminoglycan sulfotransferases are concerned in this sulfation.Regarding the glycosaminoglycan sulfotransferase which transfers asulfate group to heparin/heparan sulfate, heparan sulfateN-deacetyl/N-sulfotransferase (hereinafter sometimes referred to as“NDST”), heparan sulfate 2-O-sulfotransferase (hereinafter sometimesreferred to as “HS2ST”), heparan sulfate 3-O-sulfotransferase(hereinafter sometimes referred to as “HS3OST”), heparan sulfate6-O-sulfotransferase (hereinafter sometimes referred to as “HS6ST”) andthe like have been isolated from various organisms, particularly fromhuman, and their cDNA molecules have been cloned.

A cDNA of human HS3OST has been disclosed in J. Biol. Chem., 272,28008-28019 (1997), and the cDNA described in the reference has beenregistered at GenBank as accession number AF019386.

Although an enzyme which can transfer a sulfate group to aglycosaminoglycan having a heparin skeleton is markedly useful becauseof its high possibility to be used in the enzymatic synthesis of heparinand heparan sulfate, such an enzyme has high substrate specificity sothat it is necessary to carry out the synthesis efficiently by usingvarious types of the enzyme for the purpose of industrially synthesizingvarious types of heparin and heparan sulfate. However, it cannot be saidyet that there are sufficient variations of the enzyme capable oftransferring a sulfate group to the heparin skeleton.

Accordingly, in the case where production of a glycosaminoglycan havinga new structure becomes possible by using an enzyme, it becomes possibleto search for a physiological activity possessed by such aglycosaminoglycan.

DISCLOSURE OF THE INVENTION

An object of the invention is to provide a novel sulfotransferase andalso provide a means for obtaining the enzyme in a large amount by aconvenient method through cloning of a cDNA encoding the amino acidsequence of the polypeptide to thereby increase variations ofglycosaminoglycan which can be synthesized by enzyme chemistry and alsocontribute to the elucidation of structure-function relationship ofglycosaminoglycan having heparin skeleton.

Accordingly, the invention relates to the followings.

-   (1) A polypeptide which comprises amino acid numbers 37 to 346 in    the amino acid sequence represented by SEQ ID NO:2, or a polypeptide    of a sulfotransferase which comprises an amino acid sequence having    substitution, deletion, insertion, addition and/or transposition of    at least one amino acid in the amino acid sequence and has activity    of transferring a sulfate group from a sulfate group donor to a    glycosaminoglycan which is a sulfate group acceptor.-   (2) The polypeptide according to (1), which consists of the amino    acid sequence represented by SEQ ID NO:2.-   (3) The polypeptide according to (1), which consists of amino acid    numbers 37 to 346 in the amino acid sequence represented by SEQ ID    NO:2.-   (4) The polypeptide according to any one of (1) to (3), wherein the    glycosaminoglycan is heparin or heparan sulfate.-   (5) A sulfotransferase which comprises the polypeptide according to    any one of (1) to (4) and has activity of transferring a sulfate    group from a sulfate group donor to a glycosaminoglycan which is a    sulfate group acceptor.-   (6) A nucleic acid which encodes the polypeptide according to any    one of (1) to (4) or the sulfotransferase according to (5).-   (7) A nucleic acid which consists of the nucleotide sequence    represented by SEQ ID NO:1.-   (8) A nucleic acid which hybridizes with the nucleic acid according    to (6) or (7) or a nucleic acid consisting of a nucleotide sequence    complementary to the nucleotide sequence under stringent conditions.-   (9) An expression vector which comprises the nucleic acid according    to any one of (6) to (8).-   (10) A recombinant which comprises the expression vector according    to (9).-   (11) A recombinant which comprises a host cell into which the    expression vector according to (9) is introduced.-   (12) A process for producing a polypeptide or a sulfotransferase,    which comprises growing the recombinant according to (10) or (11),    and recovering the polypeptide according to any one of (1) to (4) or    the sulfotransferase according to (5) from the obtained grown    recombinant.-   (13) An enzyme agent for synthesizing a glycosaminoglycan comprising    the structure represented by the following formula (1), which    comprises the polypeptide according to any one of claims 1 to 4 or    the sulfotransferase according to claim 5:

-   (14) A process for producing a glycosaminoglycan comprising the    structure represented by the following formula (1), which comprises    reacting the enzyme agent according to (13) with heparin or heparan    sulfate to transfer a sulfate group from a sulfate group donor to a    sulfate group acceptor:

-   (15) Use of the polypeptide according to any one of (1) to (4) or    the sulfotransferase according to (5) as a catalyst for synthesizing    a glycosaminoglycan comprising the structure represented by the    following formula (1).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing Western blotting analysis of purifiedSFT-1-FLAG described in Examples.

FIG. 2 is a graph showing the activity of transferring a sulfate groupto heparan sulfate and heparin. The circles show the sulfate grouptransferring activity to heparan sulfate, and the squares show thesulfate group transferring activity to heparin.

FIG. 3 is a graph showing the activity of transferring a sulfate groupto chondroitin sulfate D, chondroitin, dermatan sulfate and dermatan.The open circles, the closed squares, the closed circles and the opensquares show the sulfate group transferring activity to chondroitinsulfate D, chondroitin, dermatan sulfate and dermatan, respectively.

FIG. 4 is a graph showing a radioactivity distribution in each fractionwhen a glycosaminoglycan prepared by transferring a sulfate grouplabeled with a radioisotope to heparin by using the enzyme agent of theinvention was degraded with heparin lyases, and the thus obtainedproduct was fractionated by high performance liquid chromatography. Theordinate shows radioactivity (dpm×10⁻³) of ³⁵S, and the abscissa showsretention time (min).

FIG. 5 is a graph showing a radioactivity distribution in each fractionwhen a “concentrated sample” was digested with heparin 2-sulfatase, andthe resulting product was again fractionated by high performance liquidchromatography (B). A indicates a control of not treating with heparin2-sulfatase. The ordinate shows radioactivity (dpm×10⁻³) of ³⁵S, and theabscissa shows retention time (min).

FIG. 6 is a graph showing a radioactivity distribution in each fractionwhen a sample obtained by specifically degrading only the unsaturateduronic acid of the “concentrated sample” was fractionated by highperformance liquid chromatography (A). B is a graph showing aradioactivity distribution in each fraction when a standard samplelabeled with a radioisotope was fractionated by high performance liquidchromatography in the same manner. The ordinate shows radioactivity(dpm×10⁻³) of ³⁵S or ³H, and the abscissa shows retention time (min).

FIG. 7 is a graph showing a radioactivity distribution in each fractionwhen the unsaturated disaccharide obtained by degrading heparan sulfateto which the “sulfate group labeled with a radioisotope” had beentransferred using the enzyme agent 1 of the invention was fractionatedby high performance liquid chromatography. The ordinate showsradioactivity (dpm×10⁻³) of ³⁵S, and the abscissa shows retention time(min).

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors have conducted intensive search on a DNAcomprising a nucleotide sequence encoding glycosaminoglycansulfotransferase capable of sulfating heparan sulfate and found a novelDNA having a nucleotide sequence encoding a polypeptide of the enzyme,and have confirmed that the glycosaminoglycan sulfotransferase can beobtained by expressing the DNA and that glycosaminoglycan having a newstructure can be produced by using the glycosaminoglycansulfotransferase. Thus, the invention has been accomplished.

The embodiments of the invention are described below.

(1) Enzyme of the Invention/Polypeptide of the Invention

The enzyme of the invention is a glycosaminoglycan sulfotransferase(SFT-1) which comprises a polypeptide comprising an amino acid sequenceconsisting of at least amino acid numbers 37 to 346 in the amino acidsequence represented by SEQ ID NO:2 and also has activity oftransferring a sulfate group from a sulfate group donor to aglycosaminoglycan which is a sulfate group acceptor.

Examples of the polypeptide according to the enzyme of the invention(hereinafter sometimes referred to as “polypeptide of the invention”)include a polypeptide consisting of amino acid numbers 1 to 346represented by SEQ ID NO:2 and a polypeptide consisting of an amino acidsequence consisting of amino acid numbers 37 to 346 in the amino acidsequence represented by SEQ ID NO:2. It is preferred that thepolypeptides are derived from a mammal, particularly desirably fromhuman. Among the polypeptides, a polypeptide consisting of an amino acidsequence consisting of amino acid numbers 37 to 346 which excludes thepresumed transmembrane region from the amino acid sequence representedby SEQ ID NO:2 (a region consisting of amino acid numbers 1 to 36 in SEQID NO:2) is particularly preferred since it becomes a so-calledsolubilized form which facilitates its preparation and application.

In general, it is known that the enzyme activity is maintained when oneor plural (generally from 2 to 34) constituting amino acids in an aminoacid sequence of an enzyme protein are substituted, deleted, inserted,added and/or transpositioned, so that it can regarded as a variant ofthe same enzyme, and in the case where partial mutations such assubstitution, deletion, insertion, addition and/or transposition of oneor plural (generally from 2 to 34) constituting amino acids are alsogenerated in the amino acid sequence represented by SEQ ID NO:2 of thepolypeptide of the invention, this can be regarded as a substance whichis substantially identical to the polypeptide of the invention, so longas it keeps the sulfate group transferring activity which is describedlater (such a polypeptide having partial mutations in the polypeptidecomprising the amino acid sequence represented by SEQ ID NO:2 isdescribed as “a modified polypeptide” for the sake of convenience). Itis preferred that the amino acid sequence of the modified polypeptidehas a homology of 90% or more, preferably 95% or more, more preferably97% or more, with the amino acid sequence represented by SEQ ID NO:2.The homology of the amino acid sequence can be easily calculated byusing conventionally known computer software such as FASTA, and thesoftware can also be provided by internet.

Also, in the above-described polypeptide of the invention, a saccharidechain can be linked to a constituting amino acid of the polypeptide, solong as its amino acid sequence is the same as described above and ithas the above-described enzyme activity. That is, an embodiment ofglycoprotein is included in the polypeptide of the invention as a matterof course.

The sulfate group donor used in the reaction of the enzyme of theinvention is not particularly limited, so long as it is a substancecapable of transferring the sulfate group to a glycosaminoglycan whichis a sulfate group acceptor, but 3′-phosphoadenosine 5′-phosphosulfate(active sulfate: hereinafter referred also to as “PAPS”) which isgenerally known to function as a sulfate group donor in the living bodyis preferred due to a high possibility that it is a sulfate group donorfor the enzyme of the invention in the living body.

Examples of the glycosaminoglycan as the sulfate group acceptor for theenzyme of the invention include hyaluronic acid, chondroitin,chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate,heparin and the like, glycosaminoglycan having the so-called heparinskeleton such as heparan sulfate or heparin is particularly preferred,and heparan sulfate is most particularly preferred. Also, as is alsoapparent from the following Examples, the enzyme of the invention doesnot substantially have activity of transferring a sulfate group to sharkcartilage-derived chondroitin sulfate, chondroitin obtained bydesulfating bovine bronchus-derived chondroitin sulfate, swine skinderived dermatan sulfate and desulfated dermatan sulfate prepared byremoving sulfate group from cockscomb-derived dermatan sulfate.

It is possible to easily confirm the sulfate group transferring activityof the enzyme of the invention, for example, by carrying out the enzymereaction in a buffer at a temperature of 20 to 40° C. by using PAPSlabeled with a label such as a radioisotope (³⁵S, ³H (tritium) or thelike) or a fluorescent material (a radioisotope is preferred since itdoes not generate steric hindrance in the substrate) and also by using aglycosaminoglycan which is a sulfate group acceptor, and then examiningwhether or not the acceptor is labeled with the label, for example, bychecking the reaction solution after the reaction through thecombination of a separation means such as gel filtration or highperformance liquid chromatography (hereinafter also referred to as“BPLC”) with a label-detecting means (radioactivity detecting means suchas a scintillation counter or autoradiography when a radioisotope isused as the label, or detection by a fluorescence detector when afluorescent material is used as the label).

Since the enzyme of the invention obtained in this manner has activityof specifically transferring the sulfate group to a glycosaminoglycan,particularly to heparin and heparan sulfate, it is possible to use thisas the enzyme agent of the invention which is described later.

(2) Nucleic Acid of the Invention, Expression Vector of the InventionAnd Recombinant of the Invention

The nucleic acid of the invention is a nucleic acid which encodes theenzyme of the invention or polypeptide of the invention.

The nucleic acid of the invention is not limited to deoxyribonucleicacid (DNA) or ribonucleic acid (RNA), so long as it encodes the enzymeof the invention or polypeptide of the invention, and it may be eithersingle-stranded or double-stranded. However, since the above-describedenzyme of the invention and polypeptide of the invention arehuman-derived amino acid sequences, it is preferable that it is a DNAcapable of encoding a polypeptide in many organisms including human.

The term “nucleic acid encoding” as used herein means both of a nucleicacid consisting of a nucleotide sequence complementary to the nucleotidesequence of a template chain to be used as the template for mRNAsynthesis and a nucleic acid consisting of a nucleotide sequence of thetemplate chain, generally in the transcription in protein (polypeptide)synthesis.

Examples of the nucleotide sequence of such a nucleic acid include thenucleotide sequence represented by SEQ ID NO:1, a nucleotide sequenceconsisting of nucleotide numbers 109 to 1041 (a nucleotide sequencecorresponding to the coding region of the amino acid numbers 37 to 346)in the nucleotide sequence represented by SEQ ID NO:1, and nucleotidesequences complementary to these nucleotide sequences, and nucleic acidsconsisting of such nucleotide sequences are included in the nucleic acidof the invention.

In addition, it is known that a single-stranded nucleic acid hybridizeswith a nucleic acid comprising a nucleotide sequence complementarythereto under certain conditions, and the nucleic acid of the inventionincludes nucleotide chains consisting of the nucleotide sequencerepresented by SEQ ID NO:1, a nucleotide sequence consisting ofnucleotide numbers 109 to 1041 in the nucleotide sequence represented bySEQ ID NO:1, and nucleotide sequences complementary to these nucleotidesequences under stringent conditions.

Examples of the stringent conditions include conditions of 42° C. in thepresence of 50% formamide, 5×SSPE (sodium chloride/sodium phosphate/EDTA(ethylenediaminetetraacetic acid) buffer), 5× Denhardt's solution, 0.5%SDS (sodium dodecyl sulfate) and 100 μg/ml of denatured salmon spermDNA, and under conditions substantially identical thereto. That is, thestringent conditions are conditions employed in the generalhybridization of genes and included in the term “under stringentconditions” as used herein, so long as they are conditions used in thescreening and the like which use Northern blotting, Southern blotting orhybridization.

The DNA which consists of the nucleotide sequence consisting ofnucleotide numbers 109 to 1041 in the nucleotide sequence represented bySEQ ID NO:1, which is one of the preferred illustrative embodiments ofthe nucleic acid of the invention, can be prepared by the methoddescribed in the following Examples, or since the complete nucleotidesequence thereof has been found by the invention, it can also beprepared by carrying out a polymerase chain reaction (hereinafter alsoreferred to as “PCR”) in the usual way, for example, by using ahuman-derived CDNA library as the template and using a 5′ primer (SEQ IDNO:3) and 3′ primer (SEQ ID NO:4). In the same manner, a DNA consistingof the nucleotide sequence represented by SEQ ID NO:1 can also beprepared by carrying out PCR using a primer represented by SEQ ID NO:5as the 5′ primer and a primer represented by SEQ ID NO:4 as the 3′primer.

The expression vector of the invention contains the above-describednucleic acid of the invention, which is generally constituted by thenucleic acid of the invention as a DNA and a conventionally known basalvector (a plasmid, phage, virus or the like) into which the enzyme ofthe invention is introduced, and is constructed in such a manner thatthe enzyme of the invention or polypeptide of the invention can beexpressed in a host cell.

The basal vector to be used as the above-described expression vector ofthe invention can be optionally selected by those skilled in the artaccording to the host cell used, and the expression vector of theinvention can be constructed by ligating the above-described nucleicacid of the invention to the thus selected basal vector in the usualway.

In addition, in order to facilitate its secretion, isolation,purification and analysis in preparing the enzyme of the invention orpolypeptide of the invention by expressing the expression vector of theinvention, the enzyme of the invention or polypeptide of the inventionmay be constructed in such a manner that it can be expressed as a fusionprotein with a marker peptide. In the arrangement of the enzyme of theinvention or polypeptide of the invention and the marker peptide in thiscase, the marker peptide may be bound to the C-terminal side of theenzyme of the invention or polypeptide of the invention or to theN-terminal side thereof In addition, such a binding when made via aspacer consisting of an amino acid sequence having no physiologicalactivity (a peptide consisting of about 2 to 10 amino acids) is notlimited, so long as the enzyme of the invention or polypeptide of theinvention has activity of transferring a sulfate group from a sulfategroup donor to a glycosaminoglycan which is the sulfate group acceptor.

The marker peptide means any peptide, for example, selected from thegroup consisting of a signal peptide (a peptide consisting of 15 to 30amino acid residues, which is present in the N-terminus of many proteinsand functioning intracellularly for the selection of a protein: e.g.,OmpA, OmpT, Dsb or the like), protein kinase A, protein A (a protein ofabout 42,000 in molecular weight, which is a constituting component ofStaphylococcus aureus cell wall), glutathione S transferase, His tag (asequence of 6 to 10 histidine residues), myc tag (a sequence of 13 aminoacid residues, derived from cMyc protein), FLAG peptide (a marker foranalysis consisting of a sequence of 8 amino acid residues), T7 tag (asequence of the first 11 amino acid residues of gene 10 protein), S tag(a sequence of 15 amino acid residues, derived from pancreatic RNase A),HSV tag, pel B (a sequence of 22 amino acid residues of Escherichia coliouter membrane protein pel B), HA tag (a sequence of 10 amino acidresidues, derived from hemagglutinin), Trx tag (thioredoxin sequence),CBP tag (a calmodulin binding peptide), CBD tag (a cellulose bindingdomain), CBR tag (a collagen binding domain), β-lac/blu (β-lactamase),β-gal (β-galactosidase), luc (luciferase), HP-Thio (His-patchthioredoxin), HSP (heat shock protein), Lnγ (laminin γ peptide), Fn(fibronectin partial peptide), GFP (green fluorescent peptide), YFP(yellow fluorescent peptide), CFP (cyan fluorescent peptide), BFP (bluefluorescent peptide), DsRed, DsRed2 (red fluorescent peptide), MBP(maltose binding peptide), LacZ (lactose operator), IgG (immunoglobulinG), avidin and protein G, and any one of these marker peptides can beused. Among these, signal peptide, protein kinase A, protein A,glutathione S transferase, His tag, myc tag, FLAG tag, T7 tag, S tag,HSV tag, pelB and HA tag are particularly preferred since expression ofthe enzyme of the invention and polypeptide of the invention by geneticengineering techniques and their secretion, isolation, purification andanalysis become more easy.

As the host cell into which the expression vector of the invention is tobe introduced, it is possible to use either a procaryotic cell (e.g.,Escherichia coli or the like) or a eucaryotic cell (e.g., yeast, insectcell, mammalian cell or the like). Particularly, in the case where aprocaryotic cell is used as the host cell, saccharide chain addition andthe like do not occur when the nucleic acid of the invention isexpressed, so that the peptide of the invention to which saccharidechains are not added can be obtained. However, since the enzyme of theinvention or polypeptide of the invention is an enzyme or polypeptidegenerally expressed in eucaryote, a eucaryotic cell is preferred as thehost cell, and its preferred examples include insect cells (they aresuperior in terms of the large scale synthesis of the enzyme of theinvention or polypeptide of the invention) or mammalian cells (they aresuperior in terms that they are cells in which the enzyme of theinvention is originally expressed).

The recombinant of the invention is a cell in which the vector of theinvention constructed using a suitable basal vector into such a hostcell in the usual way.

(3) Enzyme Production Method of the Invention

The enzyme production method of the invention is a method for producingthe polypeptide of the invention or enzyme of the invention, wherein therecombinant of the invention is allowed to grow, and the polypeptide ofthe invention or enzyme of the invention is recovered from the thusobtained grown material.

The method for growing the recombinant of the invention can be carriedout by those skilled in the art by optionally selecting a methodsuitable for the host cell used in the recombinant. Also, the term“grow” is a general idea of not only culturing the recombinant inoutside of a living body, for example, by using a culturing apparatus ora culturing tool, but also propagating it in the living body byadministering the host cell to the living body.

The grown material in the production method of the invention includes amedium obtained by culturing the cell in the outside of the living bodyand the cultured recombinant itself, as well as excrements, secretions,body fluids, tissues and the like of the living body obtained when therecombinant is grown in the living body.

As the recovery of the polypeptide of the invention from a grownmaterial, it is possible to separate the polypeptide of the invention,for example, by a separation means such as gel filtration or HPLC whichis based on the difference in molecular weight and a separation meanssuch as an affinity column in which a sulfate group donor (PAPS or thelike) in the enzyme reaction of the polypeptide of the invention is madeinto a solid phase, or in the case where the polypeptide of theinvention is expressed as a fusion protein with a marker peptide, by ameans for specifically adsorbing the marker peptide. For example, in thecase where FLAG peptide is used as the marker peptide, it is possible toobtain the polypeptide of the invention easily as its fusion proteinwith the FLAG peptide by using an affinity column in which an anti-FLAGantibody is made into a solid phase.

(4) Enzyme Preparation of the Invention

The enzyme agent of the invention is an “enzyme agent for the synthesisof a glycosaminoglycan which comprises a structure represented by thefollowing formula (1) and comprises the enzyme of the invention orpolypeptide of the invention”.

Also, according to this description, the symbol in the formula (1) meansthat the projecting direction of the carboxyl group against thesaccharide ring is not limited.

The “polypeptide of the invention” and the “enzyme of the invention”according to the above-described enzyme agent of the invention are “apolypeptide of an enzyme having activity of forming a glycosaminoglycancomprising the structure of above-described formula (1)(sulfotransferase activity), by transferring a sulfate group from asulfate group donor to a glycosaminoglycan which is a sulfate groupacceptor, upon heparin or heparan sulfate” and an enzyme comprising thepolypeptide, respectively, which are contained as the active componentof the enzyme agent of the invention.

In addition, the “sulfate donor” in the enzyme reaction by the enzymeagent of the invention is not particularly limited, so long as it is “asubstance capable of transferring a sulfate group to a glycosaminoglycanwhich is a sulfate group acceptor”, and PAPS which is known to generallyfunction as the sulfate group donor for the enzyme of the invention inthe living body.

Also, in addition to the active components, the “enzyme agent of theinvention” may further contain a carrier (cellulose gel, agarose gel,silica gel, glass beads or the like), a stabilizer or filler forstabilizing the same or making a pharmaceutical preparation, otherpolypeptide (e.g., a marker peptide or the like for forming a fusionprotein with the “peptide of the invention” in the case where the“polypeptide of the invention” is synthesized by genetic engineeringtechniques) or a saccharide chain (e.g., a saccharide chain is added tothe polypeptide of the invention in some cases when a eucaryote-derivedcell is used as the host in synthesizing the “enzyme of the invention”or “polypeptide of the invention” by genetic engineering techniques),without problems, so long as they do not hinder the sulfotransferaseactivity possessed by the polypeptide of the invention or enzyme of theinvention.

Such an enzyme agent of the invention can be used in a method forproducing heparin or heparan sulfate comprising the structure of thefollowing formula (1), by transferring sulfate group from a “sulfategroup donor” to a glycosaminoglycan (saccharide chain production methodof the invention).

(5) Sugar Chain Production Method of the Invention

The glycosaminoglycan obtained by the saccharide chain production methodof the invention is a “glycosaminoglycan having the heparin skeletonwhich contains a disaccharide represented by the following formula (1)as the basal skeleton”.

In addition, the above-described formula (1) is more specificallydisaccharides represented by the following formulae (3) and (4), andeither one of the disaccharides is contained at a frequency of one ormore, preferably 3 or more, and more preferably 5 or more, per moleculeof the “product by the saccharide chain production method of theinvention”.

Since the “product by the saccharide chain production method of theinvention” is prepared by allowing the above-described “enzyme agent ofthe invention” to react as the catalyst upon a glycosaminoglycan, itsweight average molecular weight is a weight average molecular weightclose to that of the heparin or heparan sulfate used as the material.For example, the weight average molecular weight of the “product by thesaccharide chain production method of the invention”, measured by gelfiltration, is from 3,000 to 30,000 Da, preferably from 4,000 to 27,000Da, and most preferably from 5,000 to 25,000 Da.

The invention is described below more illustratively based on Examples,but the invention is not limited thereto.

EXAMPLE 1 Search of Gene Data Base And Determination of NucleotideSequence of the Nucleic Acid of the Invention

Using a conventionally known human-derived heparan sulfate3-O-sulfotransferase (HS3OST) gene, analogous genes from a gene database were searched. The sequence used was SEQ ID NO:AFO19836 of theHS3OST gene. In this case, Blast [Altschul et al., J. Mol. Biol., 215,402-410 (1990)] was used in the search.

As a result, an analogous sequence was found in a genomic sequenceGeneBank Accession No. AL 355498, and a novel gene having homology withthe HS3OST gene was identified. It was estimated by a gene analyzingprogram (GENSCAN: manufactured by Stanford University) that this novelgene is encoded by two exons.

(1) Confirmation of Coding Region of the Polypeptide of the Invention

Using Human Kidney Marathon-Ready cDNA (manufactured by CLONTECH), PCR(35 cycles of 94° C. for 5 seconds and 68° C. for 4 minutes) was carriedout with the attached AP1 primer (AP1 and AP2 adapters are attached toboth ends of a cDNA fragment) and a primer set up to a sequence moietyaround the 5′-terminus of the second exon (GP-226: SEQ ID NO:6).Subsequently, nested PCR (40 cycles of 94° C. for 5 seconds and 68° C.for 4 minutes) was carried out with the AP2 primer attached to theMarathon cDNA and a primer set up to the sequence moiety (GP-224: SEQ IDNO:7). The PCR product obtained as the result was subjected to anagarose gel electrophoresis, and a band of about 450 b was recovered byusing Gel Extraction Kit (manufactured by QIAGEN). As a result of theanalysis of the nucleotide sequence of the thus obtained DNA fragment bya conventional method, a sequence of the second exon was confirmed insuccession to a sequence of the first exon (N-terminal 36 amino acidswere encoded). The sequence was the same as that predicted by the geneanalysis program. Accordingly, it was confirmed that the coding regionfor the polypeptide of the invention is the sequence shown in SEQ IDNO:1 in which the first exon and the second exon are bound to eachother.

(2) Cloning of Second Exon

Based on the above results, those which are encoded by the first exonare only the N-terminal 36 amino acids. Since the second exon encodesthe majority of the polypeptide of the invention, it was considered thatthe principal part of the enzyme including the active region iscontained in the second exon (the polypeptide of the invention encodedby the second exon is called SFT-1 for the sake of convenience).Accordingly, cloning of the second exon moiety was carried out by usinga genomic DNA as the template.

Using Human Genomic DNA (manufactured by CLONTECH) as the template, PCR(35 cycles of 94° C. for 15 seconds, 50° C. for 30 seconds and 68° C.for 1 minute) of a region containing the second exon was carried out.The primers used were set to the genomic sequences of an upstream moietyof the second exon (SFTex2F: SEQ ID NO:8) and a downstream moiety of thetermination codon (SFTex2R: SEQ ID NO:9). The thus obtained fragment ofabout 1 kb was purified in the usual way, and its nucleotide sequencewas analyzed to confirm that the sequence of the second exon wasobtained.

EXAMPLE 2 Introduction of SFT-1 Gene Into Expression Vector

In order to prepare a gene expression system, the second exon DNAobtained above was firstly introduced into an expression vector pDONR201of the Gateway system manufactured by Invitrogen, and Bacmid was furtherprepared by the Bac-to-Bac system manufactured by Invitrogen. Thedetails are explained below.

(1) Preparation of Entry Clone For the Novel Sulfotransferase

Using the PCR product obtained above by amplifying the second exon asthe template, PCT (30 cycles of 94° C. for 15 seconds and 68° C. for 3minutes) was again carried out to obtain a DNA fragment for used in theGateway system. The primers used were a 5′ primer (SFTgateF2: SEQ IDNO:10) and a 3′ primer (SFTgateRstop: SEQ ID NO:11), prepared by addinga sequence for Gateway system to a sequence close to the 5′-terminus ofthe second exon and a sequence close to the termination codon. The DNAfragment was purified in the usual way and introduced into pDONR201 by aBP clonase reaction to prepare an entry clone. The reaction was carriedout by incubating 1 μl of the DNA fragment of interest, 1 μl (150 ng) ofpDONR201, 2 μl of a reaction buffer, 4 μl ofTris-ethylenediaminetetraacetic acid (EDTA) buffer (hereinaftersometimes referred to as “TE”) and 2 μl of BP clonase mix, at 25° C. for1 hour. The reaction was terminated by adding 1 μl of proteinase K andkeeping at 37° C. for 10 minutes.

Thereafter, 5 μl of the above-described reaction solution was mixed with100 μl of competent cells (Escherichia coli DH5α) to carry outtransformation by a heat shock method, and then the cells were spread onthe LB medium containing kanamycin. A colony was picked up on the nextday and cultured in 3 ml of LB medium containing kanamycin, and thenplasmid was extracted and purified using QIAprep Spin Miniprep Kit(manufactured by QIAGEN). Using a part of the thus obtained plasmid, itsnucleotide sequence was analyzed by a conventional method to confirmthat the DNA of interest has been introduced.

(2) Preparation of Expressing Clone

The above-described entry clone has attL which is a recombination regionwhen λ phage is cut out from Escherichia coli, on both sides of itsinsertion site, and the insertion site is transferred to a destinationvector by mixing LR clonase (a mixture of recombinases Int, IHF and Xis)with the destination vector so that a expressing clone is prepared. Thespecific steps are as follows.

Firstly, 1 μl of the entry clone, 0.5 μl (75 ng) of pFBIF, 2 μl of LRreaction buffer, 4.5 μl of TE and 2 μl of LR clonase mix were allowed toreact at 25° C. for 1 hour, and the reaction was terminated by adding 1μl of proteinase K and incubating at 37° C. for 10 minutes (pFBIF-SFT-1is purified by this recombination reaction). The pFBIF is prepared byinserting Igκ signal sequence (SEQ ID NO:12) and FLAG peptide (SEQ IDNO:13) into pFastBac1, and in order to insert Gateway sequence byinserting a DNA fragment obtained by primers OT20 (SEQ ID NO:15) andOT21 (SEQ ID NO:16) using OT3 (SEQ ID NO:14) as the primer into theBamHI and EcoRI sites in the same manner as described above, aconversion cassette was inserted using Gateway Vector Conversion System(manufactured by Invitrogen). The Igκ signal sequence was inserted toconvert the expressed protein into secretion type, and the FLAG tag tofacilitate its formation.

Thereafter, 5 μl of the above-described reaction solution was mixed with50 μl of competent cells (Escherichia coli DH5α), followed bytransformation by a heat shock method, and then the cells were spread onthe LB medium containing ampicillin. A colony was picked up on the nextday and cultured in 5 ml of LB medium containing ampicillin, and thenthe plasmid (pFBIF-SFT-1) was extracted and purified using QIAprep SpinMiniprep Kit (manufactured by QIAGEN). Using a part of the thus obtainedplasmid, its nucleotide sequence was analyzed by a conventional methodto confirm that the DNA of interest has been introduced.

(3) Preparation of Bacmid By Bac-To-Bac System

Next, the sequence of SFT-1 was inserted into Bacmid capable ofmultiplying in insect cells, by carrying out recombination between theabove-described pFBIF-SFT-1 and pFastBac using the Bac-to-Bac system(manufactured by Invitrogen). This system is a system in which a gene ofinterest is incorporated into Bacmid by a recombinant protein producedfrom a helper plasmid, by simply introducing the gene ofinterest-inserted pFastBac into a Bacmid-containing Escherichia colistrain (E. coli DH10BAC) by using the recombination region of Tn7. Inaddition, since the lacZ gene is contained in Bacmid, it is possible tocarry out the classical selection based on the color of colonies (blue(no insertion)—white (insertion)).

That is, the above-described purified vector (pFBIF-SFT-1) was mixedwith 50 μl of competent cells (Escherichia coli DH10BAC), followed bytransformation by a heat shock method, the resulting cells were spreadon the LB medium containing kanamycin, gentamicin, tetracycline,5-bromoindolyl β-D-galactopyranoside (Bluo-gal) and isopropylβ-D-thiogalactopyranoside (IPTG), and then a white single colonyisolated on the next day was further cultured to recover Bacmid.

EXAMPLE 3 Introduction of Bacmid Into Insect Cell And Recovery of SFT-1

The above-described Bacmid obtained from a white colony was introducedinto an insect cell Sf21 (manufactured by Invitrogen). That is, 9×10⁵cells/2 ml of the Sf21 cells were added to Sf-900IISFM (manufactured byInvitrogen) containing antibiotics in a 35 mm dish and cultured at 27°C. for 1 hour to adhere the cells. As a solution A, 100 μl ofSf-900IISFM containing no antibiotics was added to 5 μl of Bacmid DNA.As a solution B, 100 μl of Sf-900IISFM containing no antibiotics wasadded to 6 μl of Cell FECTIN solution (manufactured by Invitrogen).Thereafter, the solution A and solution B were thoroughly mixed andincubated at room temperature for 15 to 45 minutes. After confirmingthat the cells were adhered, the culture medium was sucked and 2 ml ofSf-900IISFM containing no antibiotics was added. To a solution preparedby mixing the solution A and solution B (lipid-DNA complexes), 800 μl ofSf-900IISFM containing no antibiotics was added and thoroughly mixed.The culture medium was sucked from the cell suspension, and dilutedlipid-DNA complexes solution was added to the cells and incubated at 27°C. for 5 hours. Thereafter, the transfection mixture was removed, 2 mlof Sf-900IISFM containing antibiotics was added thereto, and then 72hours thereafter, the cells were peeled off by pipetting to recover thecells and culture medium. This was centrifuged at 1,200×g for 10minutes, and the supernatant was stored in another tube (which was usedas a first virus solution).

Into a T75 culture flask, 6×10⁶ Sf21 cells/15 ml Sf-900IISFM(manufactured by Invitrogen) (containing antibiotics) was put, 1 ml ofthe first virus solution was added thereto, followed by culturing at 27°C. for 96 hours. After the culturing, the cells were peeled off bypipetting to recover the cells and culture medium. This was centrifugedat 1,200×g for 10 minutes, and the supernatant was stored in anothertube (this was used as a second virus solution).

Furthermore, the 6×10⁶ Sf21 cells/15 ml Sf-900IISFM (manufactured byInvitrogen) (containing antibiotics) was put into a T75 culture flask,and 1 ml of the second virus solution was added thereto, followed byculturing at 27° C. for 72 hours. After the culturing, the cells werepeeled off by pipetting to recover the cells and culture medium. Themixture was centrifuged at 1,200×g for 10 minutes, and the supernatantwas stored in another tube (which was used as a third virus solution).

In addition, 100 ml of Sf21 cell suspension was put into a 100 mlcapacity spinner flask at a density of 6×10⁵ cells/ml, and 1 ml of thethird virus solution was added thereto, followed by culturing at 27° C.for about 96 hours. After the culturing, the cells and culture mediumwere recovered. The mixture was centrifuged at 1,200×g for 10 minutes,and the supernatant was recovered.

Sodium azide, sodium chloride and calcium chloride were added to 10 mlof this culture supernatant to give final concentrations of 0.05% sodiumazide, 150 mmol/l sodium chloride and 2 mmol/l calcium chloride. After50 μl of an anti-Flag antibody gel (Anti-Flag M1 monoclonal antibodyAgarose Affinity Gel, manufactured by SIGMA) was added thereto, themixture was gently stirred for 12 hours. After removing the supernatantby carrying out centrifugation (1,000×g, 3 minutes, 4° C.), washing wascarried out three times with Tris buffered saline (TBS) containing 1mmol/l calcium chloride. By removing excess washing solution by carryingout centrifugation (1,000×g, 3 minutes, 4° C.), an SFT-1-FLAG fusionprotein was obtained and used as the enzyme agent of the invention foractivity measuring use.

EXAMPLE 4 Confirmation of SFT-1-FLAG And Measurement of Enzyme Activity(1) Confirmation of SFT-1

Using 5 μl of a gel to which the fusion protein (SFT-1-FLAG) purifiedabove was bound, Western blotting was carried out in accordance with theusual method using a peroxidase-labeled anti-FLAG antibody (Anti-FLAG M2Peroxidase, manufactured by SIGMA) (FIG. 1). As a result, it wasconfirmed that the fusion protein of FLAG protein with the novelsulfotransferase expressing in the culture supernatant was recovered andpurified.

(2) Measurement of the Activity of the Enzyme Agent of the Invention ToTransfer Sulfate Group To Heparan Sulfate And Heparin

The fusion protein (SFT-1-FLAG) purified from a culture supernatant wasadded to 50 mmol/l of an imidazole-hydrochloric acid buffer (pH 6.8)containing 75 μg/ml of protamine hydrochloride, to which weresubsequently added [³⁵S]-PAPS (5×10⁵ cpm, manufactured by NEN) as thesulfate donor, and heparan sulfate (derived from bovine kidney:manufactured by Seikagaku Corporation) and heparan (derived from swineintestines: manufactured by SIGMA) (500 μmol/l as the amount ofhexosamine) as the sulfate acceptors, and the total volume was adjustedto 50 μl with distilled water. This reaction solution was allowed toreact at 37° C. for 20 minutes, and then the reaction was terminated byheating at 100° C. for 3 minutes to deactivate the enzyme. After 130 μlof ethanol containing 1.3% potassium acetate and 0.5 mmol/l EDTA wereadded thereto, followed by stirring, and then the precipitate obtainedby centrifugation was dissolved in 50 μl of distilled water. By againcarrying out the ethanol precipitation and dissolution in 50 μl ofwater, filtration was carried out through a microfilter of 0.22 μm inpore size (manufactured by Millipore), followed by separation by HPLC.The separation was carried out by using G2500PW (manufactured by Tosoh)as the column, and 0.2 mol/l sodium chloride as the mobile phase, at aflow rate of 0.6 ml/min and at a column temperature of 35° C. Theeluates from the column were recovered as fractions of every 0.3 ml, andthe radioactivity of each fraction was counted by a scintillationcounter (FIG. 2). As a result, peak of the radioactivity was detected ata position of the elution time of about 12 minutes. Since this elutiontime coincides with the elution time of heparan sulfate or heparin asthe sulfate acceptor, it was confirmed that it shows the activity oftransferring a sulfate group to these two acceptors.

In addition, when the sulfate transferring activity was measured underthe same conditions of the above-described activity measuring methodusing chondroitin sulfate D (shark cartilage origin: manufactured bySeikagaku Corporation), chondroitin (prepared by carrying out completedesulfation of bovine bronchus-derived chondroitin sulfate in accordancewith the method described in J. Am. Chem. Soc., 79, 152-153 (1957)),dermatan sulfate (swine skin origin: manufactured by SeikagakuCorporation) and desulfated dermatan sulfate (prepared by carrying outcomplete desulfation of cockscomb-derived dermatan sulfate in accordancewith the method described in J. Am. Chem. Soc., 79, 152-153 (1957)) asthe sulfate acceptors, the sulfotransferase activity was not observedupon these acceptors (FIG. 3). Accordingly, it was suggested that theSFT-1 has the specific activity for heparan sulfate and heparin.

EXAMPLE 5 Modification of Heparin By the Inventive Enzyme Agent

Sulfation reaction of heparin was carried out using the enzyme agent ofthe invention. The reaction solution contains 11 μg of protaminehydrochloride, 0.3 mg of heparin (manufactured by SIGMA), [³⁵S]-PAPS(2.3×10⁷ dpm: manufactured by Perkin Elmer) and the enzyme agent 1 ofthe invention (20 μl) in 150 μl of 50 mM imidazole-hydrochloric acidbuffer (pH 6.8). After incubation at 37° C. for 3 hours, heparin wasrecovered by carrying out 70% ethanol precipitation twice. The mixturewas allowed to stand at room temperature to evaporate ethanol, dissolvedin 30 μl of a buffer for heparin degrading enzyme reaction (20 mM sodiumacetate buffer (pH 7.0: contains 2 mM calcium acetate)) and thenincubated at 37° C. for 2 hours by adding heparin degrading enzymes(heparinase 150 mU (manufactured by Seikagaku Corporation), heparitinaseI 90 mU (manufactured by Seikagaku Corporation) and heparitinase II 60mU (manufactured by Seikagaku Corporation): these enzymes form anunsaturated disaccharide (ΔHexA1,4GlcN) in which unsaturated uronic acid(ΔHexA) and GlcN are 1,4-glycoside-bound, by hydrolyzing the β1,4-glucoside binding moiety (GlcNβ1,4HexA) of GlcN and uronic acid(HexA) in the heparin skeleton). Thereafter, the reaction mixture washeated at 100° C. for 1 minute to terminate the reaction, filteredthrough a microfilter of 0.22 μm in pore size (manufactured byMillipore) and then separated by HPLC. The elution was carried out usingCarboPac PA1 (4×250 mm: manufactured by Dionex) and CarboPac PA1 guardcolumn (manufactured by Dionex) as the column and at a flow rate of 0.8m/min and at a column temperature of 40° C., while effecting1-6-19-38-70-76-76% of density gradient with 3 mol/l lithium chlorideagainst 0-5-8-15-20-28-40 minutes of elution time. The eluate wasfractioned at 0.2 ml, and 10 μl thereof was analyzed by a scintillationcounter to confirm eluted position of the radioactivity (FIG. 4). As aresult, a peak having strong radioactivity was found at a retention timeof 30 minutes.

Accordingly, the peak appeared at a retention time of 30 minutes wasrecovered and desalted using Cellulofine G25sf column (1×24 cm:manufactured by Seikagaku Corporation). The thus desalted sample wasconcentrated to 0.1 ml by a freeze-dryer and used as a “concentratedsample”, and 2 μl of the “concentrated sample” was digested withΔ4,5-glucuronate-2-sulfatase (an enzyme which performs desulfation byspecifically hydrolyzing the 2-position sulfuric acid ester ofunsaturated uronic acid residue: purified in accordance with the methoddescribed in Eur. J. Biochem., 145, 607-615 (1984)). As the reactionsolution, 5 ml of 20 mmol/l sodium acetate buffer (pH 6.5: contains0.15% bovine serum albumin and 4.1 mU of heparin 2-sulfatase) was used.After the reaction at 37° C. for 2 hours, the reaction was terminated byheating at 100° C. for 1 minute. After 18 μl of distilled water wasadded thereto, the mixture was filtered through a filter of 0.22 μm inpore size (manufactured by Millipore) and then separated by HPLC underthe same conditions as described above (FIG. 5). As a result, a peak wasfound at a retention time of about 30.5 minutes in the control which wasnot digested with heparin 2-sulfatase (FIG. 5A), but retention time ofthe peak was shifted to about 22 minutes in the concentrated sampletreated with Δ4,5-glucuronate-2-sulfatase (FIG. 5B). That is, sinceshifting of the peak was observed by the treatment with the enzyme whichspecifically performs desulfation of the 2-position sulfate group ofunsaturated uronic acid, it was confirmed that the unsaturateddisaccharide contained in the concentrated sample contains a ΔHexA(2S)structure of the following formula (5).

Next, 2 μl of the above-described “concentrated sample” was mixed with 2μl of 70 mM mercury acetate (pH 5.0) and allowed to stand at roomtemperature for 10 minutes to remove the unsaturated uronic acid (theunsaturated uronic acid alone in the unsaturated disaccharide isspecifically degraded by this reaction: Biochem. J. 245 795-804 (1987)),further mixed with 2 μl of 1 mol/l sodium carbonate buffer (pH 9.0) and2 μl of 0.5 mol/l sodium tetrahydroborate (0.1 mol/l sodium hydroxidesolution) and incubated at 50° C. for 30 minutes to carry out reductionreaction, and then separated by HPLC under the same conditions asdescribed above (FIG. 6). When the elution pattern of the “concentratedsample” after degradation of the unsaturated uronic acid (FIG. 6A) wascompared with the elution pattern of the standard sample labeled by thereduction reaction with sodium [³H]tetrahydroborate (GlcN(NS,3S): peak 1around 14 minutes: (GlcN(NS,3S,6S): peak 2 around 22 to 23 minutes)(FIG. 6B), retention time of the sample coincided with that of(GlcN(NS,3S,6S) (peak 2). Based on this, it was found that the“concentrated sample” after degradation of the unsaturated uronic acidis GlcN in which the 2-position amino group, the 3-position hydroxylgroup and the 6-position hydroxyl group are sulfated.

From the results of FIG. 5 and FIG. 6, it was confirmed that theunsaturated disaccharide contained in the concentrated sample is anunsaturated disaccharide (ΔHexA(2S)-GlcN(NS,3S,6S)) shown by thefollowing formula (6), and it was shown that the structure representedby the above-described formula (1) is contained in the glycosaminoglycanbefore degradation with heparin degrading enzymes.

When heparan sulfate (manufactured by SIGMA) was sulfated using theenzyme agent of the invention under the same sulfation reactionconditions as described above and digested with heparin degradingenzymes in the same manner as described above, and the thus obtainedsample was separated by HPLC, a peak was detected at a retention time ofabout 30.5 minutes (the peak shown by an arrow in FIG. 7), though theformed amount was smaller than the case of heparin. Since this is thesame retention time of ΔHexA(2S)-GlcN(NS,3S,6S) confirmed by heparin, itwas found that the glycosaminoglycan containing the structurerepresented by the above-described formula (1) was also formed whenheparan sulfate was used as the sulfate group acceptor.

INDUSTRIAL APPLICABILITY

A nucleic acid comprising a nucleotide sequence encoding a polypeptideof a novel heparan sulfate sulfotransferase capable of selectivelytransferring sulfate group to heparan sulfate is obtained by theinvention. Furthermore, a polypeptide expressed by the nucleic acid isobtained.

Also, since a nucleic acid comprising a nucleotide sequence encoding apolypeptide of a novel heparan sulfate sulfotransferase was obtained bythe invention, it is expected that the enzyme can be mass-produced to anindustrially applicable level. Furthermore, a glycosaminoglycan having anew structure is provided based on the enzyme activity possessed by theenzyme.

1-5. (canceled)
 6. A nucleic acid selected from the group consisting of(I), (II) and (III): (I) a nucleic acid which encodes: a polypeptidewhich comprises amino acid numbers 37 to 346 in the amino acid sequenceof SEQ ID NO:2, or a sulfotransferase which comprises a polypeptidewhich comprises amino acid numbers 37 to 346 in the amino acid sequenceof SEQ ID NO:2 and has activity of transferring a sulfate group from asulfate group donor to a glycosaminoglycan which is a sulfate groupacceptor; (II) a nucleic acid which consists of the nucleotide sequenceof SEQ ID NO:1 and (III) a nucleic acid which hybridizes, understringent conditions, with: the nucleic acid according to (I) or (II) ora nucleic acid which consists of the nucleotide sequence of SEQ ID NO:1or a nucleic acid consisting of a nucleotide sequence complementary tothe nucleotide sequence of the nucleic acid according to (I) or (II) orthe nucleotide sequence of SEQ ID NO:1.
 7. (canceled)
 8. (canceled) 9.An expression vector which comprises the nucleic acid according to claim6.
 10. A recombinant which comprises the expression vector according toclaim
 9. 11. A recombinant which comprises a host cell into which theexpression vector according to claim 9 is introduced.
 12. A process forproducing a polypeptide or a sulfotransferase, which comprises: growinga recombinant which comprises the expression vector according to claim10 or 11 or a recombinant which comprises a host cell into which theexpression vector according to claim 9 is introduced, and recovering apolypeptide which comprises amino acid numbers 37 to 346 in the aminoacid sequence of SEQ ID NO:2 or a sulfotransferase which comprises thepolypeptide and has activity of transferring a sulfate group from asulfate group donor to a glycosaminoglycan which is a sulfate groupacceptor from the obtained grown recombinant.
 13. (canceled)
 14. Aprocess for producing a glycosaminoglycan comprising the structure ofthe following formula (1), which comprises reacting an enzyme agent withheparin or heparan sulfate to transfer a sulfate group from a sulfategroup donor to a sulfate group acceptor:

wherein the enzyme agent comprises a polypeptide which comprises aminoacid numbers 37 to 346 in the amino acid sequence of SEQ ID NO:2, or asulfotransferase which comprises the polypeptide and has activity oftransferring a sulfate group from a sulfate group donor to aglycosaminoglycan which is a sulfate group acceptor.
 15. (canceled)